Podocalyxin (PODXL) is a transmembrane glycoprotein originally identified in kidney podocytes. Whereas normal pancreas is devoid of PODXL, 44-69% of pancreatic adenocarcinomas (PDACs) are PODXL positive. Importantly, PODXL overexpression is associated with an aggressive tumor phenotype in pancreatic cancer and several other cancer types. Yet, the mechanisms by which PODXL alters cell mechanics and promotes cancer metastasis are poorly characterized. Most of our knowledge on the patho/physiological role of PODXL has resulted from studies in Madin Darby canine kidney (MDCK) and Chinese Hamster Ovary cells. Although the cytoplasmic tail of ectopically expressed PODXL in these cells associates with cortactin, ezrin and the ezrin-radixin-moesin-binding phosphoprotein 50, none of these proteins is present in a complex with endogenous PODXL in human pancreatic cancer cells. In contrast, we have discovered a novel association of PODXL with Molecules Interacting with CasL1 (MICAL1). This discrepancy may be due to species differences and/or forced versus endogenous PODXL expression. The association of PODXL with MICAL1, which binds to F-actin, represents a novel mechanism by which PODXL may regulate cell compliance, migration and metastasis. Moreover, our compelling data provide the first evidence for the involvement of Fyn kinase and Wnt11 in PODXL-dependent signaling events. Using sophisticated bioengineering, microtechnology, imaging and molecular biology tools, we will test the hypothesis that PODXL confers chemoresistance, and promotes pancreatic cancer cell migration, invasion and metastasis via induction of cytoskeletal remodeling. In Aim 1, we propose to establish that PODXL, via its association with MICAL1, regulates cell mechanics and migration through unconfined and physically constricted microenvironments, and decipher its downstream targets. Because cell compliance correlates with invasion and metastasis, we will evaluate the role of PODXL and its downstream effectors in cell cytoplasmic stiffness and invasion through collagen type-I filled microchannels (Aim 2a). In Aim 2b, we propose to demonstrate that PODXL confers resistance to the chemotherapeutic gemcitabine drug through MICAL1-dependent activation of the Src family kinase Fyn. Because ongoing clinical trials aim to evaluate the efficacy of combining gemcitabine with dasatinib (a Src family kinase inhibitor) in pancreatic cancer and other solid tumors, we propose to establish PODXL as a predictive biomarker, which identifies subpopulations of patients who will most likely benefit from this combined treatment. In Aim 3a, we will establish the role of PODXL and its downstream effectors in pancreatic cancer chemoresistance and metastasis in vivo. Because PODXL is overexpressed in PDACs we will evaluate the potential of radiolabeled anti-PODXL antibodies for imaging primary and metastatic tumors in vivo using single photon emission computed tomography (Aim 3b). Taken together, our work will define the role of PODXL in pancreatic cancer cell mechanics, metastasis and imaging.